Monoterpene synthases (MTSs) catalyze the biosynthesis of monoterpenes, which not only play a pivotal role in plant-plant communications and are used as key ingredients for many industrial applications including flavor additives, fragrance, and biofuels. MTSs control challenging reactions that involve highly reactive carbocations in a selective and stereospecific manner. Using two well-characterized MTSs as models, (4S)-(-)-limonene synthase (LMNS) and (+)-bornyl diphosphate synthase (BPPS), we implemented an iterative approach that involves comparative atomistic simulations and experimental testing of wild-type enzymes and selected variants to identify the mechanistic underpinnings of selectivity. Free energy calculations using force-field- and hybrid quantum mechanics / molecular mechanics (QM/MM)-based molecular dynamics simulations indicated for the first time that a common reaction intermediate, the α-terpinyl cation (ATC), preferentially adopts one of two different conformations in LMNS and BPPS, thus leading to the formation of monocyclic monoterpenes in the former and bicyclic products in the latter. An assessment of the ATC absolute binding free energy in LMNS, BPPS, and selected variants revealed that non-bonded interactions with active site residues explain the propensity of the ATC to assume a favored conformation that is consistent with the experimentally determined reaction outcome. The integration of atomistic simulations with experimental validation also allowed us to assign specific functions to active site residues for controlling the ratio of monocyclic to bicyclic products in model MTSs.

Understanding catalytic mechanisms of monoterpene synthases

ACS Spring 2022

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